The hedgehog (Hh) gene was first identified during a search for embryonic lethal mutants of Drosophila melanogaster, which found that mutation of Hh resulted in altered segment patterning of the larva (Nusslein-Volhard, C.; Wieschaus, E. Nature 1980, 287, 795-801). Subsequently the gene was identified in many other invertebrates and vertebrates, including humans. Three mammalian counterparts of the Hh gene, termed Sonic hedgehog (Shh), Dessert hedgehog (Dhh), and Indian hedgehog (Ihh), were identified by combined screening of mouse genomic and cDNA libraries (Echelard, Y.; Epstein, D. J.; et al., Cell 1993, 75, 1417-1430). Hh undergoes multiple processing events, including autocatalytic cleavage of the C-terminal domain combined with addition of a cholesterol moiety at the cleavage site, and an N-terminal palmitoylation, to generate the active ligand (Lee, J. J.; Ekker, S. C.; et al., Science 1994, 266, 1528-1537; Porter, J. A.; Young, K. E.; et al., Science 1996, 274, 255-259; Pepinsky, R. B.; Zeng, C. et al., J. Biol. Chem. 1998, 273, 14037-14045).
The receptor of secreted Hh protein is a 12-transmembrane protein Patched (Ptch). Of the two vertebrate homologues of Ptch (Ptch1 and Ptch2), the role of Ptch1 is better understood. In the absence of Hh ligand, Ptch inhibits the activity of the downstream effector Smoothened (Smo). The binding of Hh inactivates Ptch, resulting in activation of Smo (Stone, D. M.; Hynes, M.; et al., Nature 1996, 684, 129-134). These proteins modulate the function of Gli (Ci in Drosophila), the only transcription factor identified to date that operates directly downstream of Hh and translocates into the nucleus where they control transcription of target genes. Gli has been shown to affect transcription of Hh pathway inhibitors such as Ptc and Hip1 in a negative feedback loop indicating that a tight control of the Hh pathway activity is required for proper cellular differentiation and organ formation.
Hh genes have the ability to induce tissue proliferation. This function is important in embryogenesis and tissue maintenance, but inappropriate activation of the pathway can result in tumorigenesis (Hunter, T. Cell 1997, 88, 333-346). Tumors in about 25% of all cancer deaths are estimated to involve aberrant Hh pathway activation. Tumorigenesis or tumor growth can result from abnormal up-regulation of Hh ligand or from deregulation of the expression or function of downstream components by, for example, loss of Ptch, activating mutations of Smo (Xie, J.; Murone, M.; et al., Nature 1998, 391, 90-92), loss of SuFu, amplification or chromosomal translocation of Gli1 or Gli2 gene amplification or stabilization of Gli2 protein (Bhatia, N.; Thiyagarajan, S.; J. Biol. Chem. 2006, 281, 19320-19326).
Critical roles of HH-GLi signaling have been implicated in a large number of human cancers (reviewed in Teglund S and Toftgard R. Biochimica et Biophysica acta 2010, 1805, 181-208): from familial basal-cell carcinomas to sporadic basal-cell carcinomas, medulloblastomas, prostate, lung, pancreas, breast and colon cancers, as well as gliomas, leukemias, lymphomas and melanomas. HH-GLi not only controls the growth of the bulk of the tumor by promoting cell survival and proliferation, but it is also required for cancer stem cell self-renewal in gliomas, leukemias and colon cancers. For example, inhibition of HH-GLI activity in epithelial cells through RNA interference (RNAi) in human carcinomas in vitro and in mouse xenografts leads to tumor disappearance, inhibition of metastatic growth and tumor recurrence.
For example, Hh has also been shown to be an early and late mediator of pancreatic cancer tumorigenesis. Shh was not detected in normal adult human pancreata but was aberrantly expressed in 70% of pancreatic adenocarcinoma specimens (Thayer, S. P.; di Magliano, M. P.; et al., Nature 2003, 425, 851-856). Participation of Shh signaling has been indicated at multiple stages of pancreatic carcinogenesis and is accompanied by multiple oncogenic factors, including K-Ras, one of the most frequently mutated genes in pancreatic cancer (Morton, J. P.; Mongeau, M. E.; et al., Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5103-5108; Ji, Z.; Mei, F. C.; et al., J. Biol. Chem. 2007, 282, 14048-14055). Activated Hh signaling was detected in cell lines established from primary and metastatic pancreatic adenocarcinomas, and the Smo inhibitor cyclopamine induced apoptosis in a subset of the pancreatic cancer cell lines both in culture and in mice (Sheng, T.; Li, C.; et al., Mol. Cancer 2004, 3, 29).
The aberrant activation of Hh-Gli signaling in several cancers has made it an attractive target for anticancer drug discovery. A variety of small molecule inhibitors of hedgehog signaling pathway have been reported (Peukert S.; Miller-Moslin K. Annual Rep. Med. Chem. 2009, 44, 323-337; Heretsch P.; Tzagkaroulaki L.; Giannis A. Bioorg. Med. Chem. 2010, 18, 6613-6624). Among them, a few candidates have been advanced into clinical trials at various stages (Mas C.; Altaba, R. I. Biochem. Pharm. 2010, 80, 712-723). Despite with these ongoing exciting efforts, there still remains a need for potent and safe inhibitors of the hedgehog signaling pathway given of the emerging drug resistance being identified as well as the critical role of Hh pathway in embryonic development.